The essential role of microRNA 146a and Forkhead box protein 3 in pathogenesis of nonsegmental vitiligo

There is a paucity of data about the impact of systemic statins on vitiliginous lesions in non-segmental vitiligo (NSV) patients. To the best of our knowledge, no other studies have considered the correlation between lipid disturbances in vitiligo and vitiligo disease activity (VIDA) score. We sought in this study to evaluate the influence of simvastatin on vitiliginous lesions in NSV patients with dyslipidemia and study the correlation between VIDA score and lipid profile. This clinical trial started with 120 patients with NSV, 79 patients had dyslipidemia and received simvastatin 80 mg daily (till normalization of lipid profile or for 4months, which came first) and only 63 patients continued till the end of the study. Lipid profile, vitiligo area severity index and VIDA were assessed before and 6months after the end of simvastatin use. Serum total cholesterol (TC), triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein, and LDL/HDL ratio showed statistically significant increases in the NSV than in the control group (p˂0.001). There was a statistically significant positive correlation between VIDA and serum levels of TC and LDL and with LDL/HDL ratio. Simvastatin significantly improved the lipid profile and significantly decreased VIDA (p < 0.011). Negative moderate correlation was found between the decrease in VIDA and duration of disease (r=-0.562, p < 0.001). Simvastatin 80 mg daily could be a helpful treatment for NSV patients with dyslipidemia, controlling the vitiligo activity and protecting against the hazardous effects of dyslipidemia. Better results can be obtained in patients with short duration of the disease.

O252 Aims: The transcription factor Foxp3 has been identified as the key molecule controlling the development and function of regulatory CD25+ T-cells. In human peripheral T-cells, the induction of Foxp3 is dependent on IL-2, and therefore immunosuppressive drugs that affect IL-2 production may hinder the development or function of regulatory T-cells. Methods: To understand the biology of Foxp3 in allogeneic reactions and to define the effect of immunosuppressive drugs on Foxp3 expression, we measured mRNA expression levels in a mixed lymphocyte reaction (MLR) in the presence and absence of the calcineurin inhibitors (CNI): Tac (10 ng/ml) and CsA (100 ng/ml), anti-CD25 mAb (5 μg/ml), and Rapa (10 ng/ml). Furthermore, to assess whether Foxp3 is operational after transplantation in patients, we analyzed Foxp3, IL-2 and TCR mRNA expression levels in endomyocardial biopsies (EMB) from cardiac allograft recipients treated with anti-CD25 mAb (N=26) or with control placebo (N=31), and in specimens (N=17) before, during and after acute rejection by Q-PCR. Results: Foxp3 mRNA expression was significantly induced after in vitro allo-stimulation. Kinetic examination of the MLR showed a 20-fold higher Foxp3 mRNA expression after 5 days of culture. Both calcineurin inhibitors CsA and Tac, and anti-CD25 mAb significantly inhibited the in vitro induced Foxp3 gene transcription (range 70-90%), whereas Rapa did not inhibit but rather delayed the induction. After clinical heart transplantation, during anti-CD25 mAb treatment, significantly lower intragraft Foxp3 mRNA transcription levels were found compared to the levels measured in EMB from placebo treated patients (2- fold, p=0.02). The low Foxp3 expression levels were accompanied by low IL-2 and TCR mRNA levels (p<0.01 and p=0.05, respectively). However, the highest Foxp3 expression levels were measured during acute rejection in the placebo group (p=0.01), that again correlated with IL-2 and TCR mRNA expression levels (p=0.01 and p<0.001, respectively). Conclusions: The high Foxp3 level during allogeneic responses suggests that regulatory activities of CD25+ T-cells or the generation of these cells is an intrinsic part of activation. CNI and anti-CD25 mAb in contrast to Rapamycin, do interfere with this immunosuppressive counter-mechanism and as a result have an inhibitory effect to tolerance induction after transplantation.

To observe the effects of fermented white mustard seed plaster on the balance of T helper cell (Th) 1/Th2, Th17/T regulatory cell (Treg) immune balance in bronchial asthma (BA) rats, so as to explore the possible mechanism of fermented white mustard seed plaster in the treatment of BA. Healthy SD rats were randomly divided into blank, model, medication and acupoint application groups, with 10 rats in each group. BA model was prepared using ovalbumin sensitization and atomization. Rats in the acupoint application group were treated with fermented white mustard seed plaster at "Dazhui" (GV14), bilateral "Feishu" (BL13) and "Fengmen" (BL12) for 4 h;rats in the medication group were given intraperitoneal injection of 1 mg/kg dexamethasone sodium phosphate;both groups were treated once daily for 14 consecutive days. Behavioral observations and scoring were conducted on rats in each group before and after intervention. HE staining was performed to observe the pathological morphological changes of lung tissue. ELISA was used to detect the contents of interferon γ (IFN-γ), interleukin (IL)-4, IL-17, IL-10, tumor necrosis factor α (TNF-α), and immunoglobulin E (IgE) in serum. real-time quantitive PCR was used to detect the mRNA levels of T-box transcription factor (T-bet), GATA binding protein 3 (GATA-3), retinoic acid-related orphan receptor γt (RORγt), and forkhead box protein 3 (Foxp3) in lung tissue. Western blot was used to detect the protein expression levels of T-bet, GATA-3, RORγt, and Foxp3 in lung tissue. After treatment, compared with the blank group, the behavioral score was significantly increased (P<0.01);the thickness of bronchial wall muscle layer increased with a large amount of inflammatory cell infiltration, thickening of alveolar wall, abnormal lung tissue structure, and severe lung consolidation;the contents of serum IL-4, IL-17, TNF-α, and IgE were significantly increased (P<0.01), while the contents of serum IFN-γ and IL-10 were significantly decreased (P<0.01);the mRNA and protein expression levels of GATA-3 and RORγt in lung tissue were increased (P<0.01), while the mRNA and protein expression levels of T-bet and Foxp3 in lung tissue were decreased (P<0.01) of rats in the model group. Compared with the model group, the behavioral score was significantly decreased (P<0.01);the pathological morphological damage of lung tissue was significantly improved;the contents of serum IL-4, IL-17, TNF-α, and IgE were significantly decreased (P<0.01, P<0.05), while the contents of serum IFN-γ and IL-10 were significantly increased (P<0.01, P<0.05);the mRNA and protein expression levels of T-bet and Foxp3 in lung tissue were increased (P<0.01), while the mRNA and protein expression levels of GATA-3 and RORγt in lung tissue were decreased (P<0.01) of rats in the medication and acupoint application groups. Compared with the medication group, the contents of serum IL-4 were significantly increased (P<0.05);the mRNA and protein expression levels of GATA-3 and RORγt in lung tissue were significantly increased (P<0.01, P<0.05), while the mRNA and protein expression levels of T-bet and Foxp3 were significantly decreased (P<0.01, P<0.05) of rats in the acupoint application group. Fermented white mustard seed plaster can alleviate airway inflammation in BA, and its mechanism may be achieved by up-regulating the expression of T-bet and Foxp3, down-regulating the expression of GATA-3 and RORγt, promoting Th1/Th2, Th17/Treg immune balance, and reducing the secretion of related pro-inflammatory factors to exert a therapeutic effect on asthma.

CD4+CD25+ Regulatory T Cells in Patients with Acute Lymphocytic Leukemia.

Helper T cells 17 (Th17) and regulatory T cells (Treg), as CD4+T lymphocyte subsets, play an important role in the process of atherosclerosis. However, there are few studies on the regulation and efficacy of atorvastatin combined with amlodipine on Th17/Treg balance in hypertension combined with carotid atherosclerosis. Therefore, this study aims to verify the efficacy and immunomodulatory effects of atorvastatin combined with amlodipine in the treatment of hypertension combined with carotid atherosclerosis. A total of 260 patients with hypertension and carotid atherosclerosis were randomly divided into atorvastatin or combined treatment group. Inflammatory factors and Th17 and Treg levels were detected by enzyme-linked immunosorbent assay and flow cytometry. The messenger ribonucleic acid expression of retinoic acid receptor-related orphan receptor gamma and forkhead spiral transcription factor were detected by real-time quantitative polymerse chain reaction. We found that the total effective rate in the treatment group was significantly higher than that in the control group. The levels of whole blood high shear viscosity, whole blood low shear viscosity, plasma specific viscosity and fibrin content in the 2 groups were significantly decreased after treatment, and the combined group was significantly lower than the control group (all P < .05). The improvement of endothelial function in the treatment group was also significantly higher than that in the control group (all P < .05). In addition, we found that there were statistically significant differences in Th17 percentage, Treg percentage and Treg/Th17 between the treatment group and the control group (P < .05). The messenger ribonucleic acid levels of retinoic acid receptor-related orphan receptor gamma and forkhead spiral transcription factor showed the same trend. Further detection of Th17-related inflammatory factors showed that the expression of interleukin (IL)-17, IL-6, IL-23 and tumor necrosis factor-α in the treatment group was significantly decreased, which was better than that in the control group (all P < .05). These data indicate that amlodipine combined with atorvastatin can improve Th17/Treg imbalance, vascular endothelial function and efficacy in patients with hypertension and atherosclerosis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential factor in the growth and maturation of blood cells as well as modulation of the immune system. Few studies have investigated its involvement in the development of vitiligo, and no studies have been performed on Egyptian patients. To assess GM-CSF serum level among non-segmental Egyptian vitiligo patients and to determine its possible role in the etiopathogenesis of the disease. Forty patients with non-segmental vitiligo and 40 age- and sex-matched subjects were assessed for levels of GM-CSF in serum using the ELISA technique. The patients in this study showed lowerlevels ofGM-CSF in serum compared to controls (mean ± SD was 33.4 ± 5.7 pg/ml versus 63.4 ± 7.4 pg/ml, respectively, p = 0.0001). No appreciable relation was detected between levels of GM-CSF in serum and age, sex, family history, and stressful events or disease activity, type, and extent, p ˃ 0.05. GM-CSF serum level may be one of the determinants of the autoimmune hypothesis in the etiopathogenesis of non-segmental vitiligo.

Skewing of responses towards T helper (Th) 17 and away from T regulatory cells (T-regs) has been suggested to be partially involved in autoimmune diseases like vitiligo. Clarify the possible role and relationship between Th17 and T-regs in vitiligo by measuring tissue, systemic levels of interleukin (IL)-17, IL-22 and Forkhead box P3. 84 non-segmental vitiligo patients and 80 controls were included. Vitiligo Area Scoring Index, Vitiligo Disease Activity and stress score were determined. Skin biopsies underwent immunohistochemical staining for IL-17, IL-22 and FoxP3 and their systemic levels were determined by ELISA and quantitative real time PCR. Mean area % of +ve immunostaining and serum levels of IL-17 (34.12 ± 5.12, 23.62 ± 8.17 pg/mL) and IL-22 (48.63 ± 19.23, 43.53 ± 11.95 pg/mL) were significantly higher in patients compared to controls (15.33 ± 4.19, 12.83 ± 3.29 pg/mL) (13.44 ± 3.82, 9.92 ± 4.7 pg/mL) (P<0.001). Mean area % of +ve immunostaining and peripheral blood levels of FoxP3 were significantly lower in patients (2.67 ± 0.54, 0.574 ± 0.32) compared to controls (7.12 ± 0.18, 1.48 ± 0.49) (P<0.001). In patients, a positive correlation between IL-17 and IL-22 was detected (r = 0.671, P<0.001), each showing negative correlation with FoxP3 (r = -0.548, P<0.001), (r = -0.382, P<0.001). VASI, VIDA and stress score correlated positively with IL-17, IL-22 and negatively with FoxP3. Th17 and T-regs are intertwined in the complexity of vitiligo giving hope of treatment through adjuvant therapies controlling the delicate balance between them.

Interferons (IFNs) are related to autoimmune responses. IFN-epsilon (IFNE) is included in IFN family, and may modulate immunological functions. Inflammation modulating functions of IFNE may be related with the pathophysiology of vitiligo. To investigate the association of nonsense polymorphism (rs2039381, Gln71Stop) of interferon-ε (IFNE) and susceptibility to vitiligo, we conducted a case-control association study in 265 non-segmental vitiligo (NSV) patients and 320 healthy controls. The nonsense single nucleotide polymorphism (SNP) (rs2039381, Gln71Stop) of IFNE was genotyped by direct sequencing. Multiple logistic regression models (log-additive, dominant, and recessive models) were applied to determine odds ratios (OR), 95% confidence interval (CI), and p values. The rs2039381 (Gln71Stop) of IFNE did not show significant differences between NSV patient group and control group. However, we found that in childhood onset NSV groups, the IFNE nonsense polymorphism (rs2039381, Gln71Stop) showed a significant association. There was significantly different distribution of nonsense polymorphism of rs2039381 (Gln71Stop) of IFNE between NSV patients (childhood <18 years) and control subjects. This study suggests that rs2039381 (Gln71Stop) polymorphism of IFNE may be related to onset time of vitiligo in NSV patients.

Expression of Regulatory γδ T Cells in Patients with Acute Graft-Versus-Host Disease

miRNAs are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3’ UTR of specific mRNA for degradation or translational repression. miRNAs have been shown to be promising biomarkers for different diseases. At present, the expression and function of miRNAs in human skin is largely unknown. The aim of the present study was to detect the differentially expressed miRNAs in non-segmental vitiligo (NSV) patients and to explore the potential role of these miRNAs in vitiligo pathogenesis. We performed the whole miRNA profiling of lesional as well as non-lesional skin from four NSV patients and four healthy skin samples from controls using TaqMan® Low Density Array. Our results suggest that 38 miRNAs were differentially expressed in the skin of patients compared to controls. We identified 13 miRNAs which were significantly differentially expressed in lesional skin of patients compared to healthy control skin. Further, 29 miRNAs were found to be significantly differentially expressed between non-lesional skin of patients and healthy control skin. Interestingly, three miRNAs were specifically down-regulated in the lesional skin compared to non-lesional skin from patients with NSV. In conclusion, for the first time the present study suggests the crucial role of differentially expressed miRNAs in NSV patients from Gujarat.

Characterization of Regulatory T Cell Subsets in Atopic Eczema

This study was aimed to compare the expressions of specific transcription factors of CD4(+) T cell subset ( T-bet, GATA-3, RORγt and FoxP3 mRNA) in peripheral blood of patients with aplastic anemia(AA), myelodysplastic syndrome(MDS), and acute myeloid leukemia(AML), and investigate their immune status and pathogenesis, so as to provide experimental basis for the choice of clinical treatment. The expression of T-box (T-bet), GATA-3, ROR-γt and Foxp3 mRNA in PBMNC were examined by RT-PCR in 42 cases of MDS, including 22 refractory anemia(MDS-RA) and 20 refractory anemia with excess blasts (MDS-RAEB), in 23 cases of AA, 17 cases of AML patients and 16 healthy volunteers respectively. The results indicated that, compared with normal control group, expressions of T-bet and RORγt mRNA in AA patient group were significantly higher (P < 0.01), expression levels of GATA3 Foxp3 mRNA were lower (both P < 0.01). There was no significant difference in expression of T-bet and GATA3 mRNA between MDS group and normal control group, but the expression levels of Foxp3 and RORγt mRNA were higher than those in normal controls (P < 0.05); T-bet and RORγt in MDS-RA group were higher than those in the normal controls(P < 0.01), and GATA3 expression significantly reduced (P < 0.05), however, there was no significant difference in expression of Foxp3 between MDS-RA and the controls. Expression levels of T-bet and RORγt mRNA in patients with MDS-RAEB and AML were lower than those in normal controls (P < 0.05), but the expression levels of GATA3 and Foxp3 mRNA were significantly higher than those in normal controls (P < 0.01). It is concluded that the transcription factor expressions are different in PBMNC of patients among these three diseases. Immune-mediated excessive apoptosis may play an important role in pathogenesis, bone marrow failure in patients with AA and MDS-RA, and abnormal clones of immature cells may be one of main reasons for bone marrow failure in AML and late stage of MDS.

Objective To investigate the expression of forkhead/winged helix transcripition factor 3 (Foxp3) in colorectal cancer and the effect of Foxp3 short hairpin RNA (shRNA) on apoptosis of colorectal cancer cells. Methods Western blotting was used to detect the expression level of Foxp3 in colorectal cancer tissues and corresponding paracancerous tissues. The expression of Foxp3 in human colorectal cancer cells HCT116, SW480, RKO and human intestinal mucosal cell line CCC-HIE-2 was detected by Western blotting. Transfected Foxp3 shRNA1, Foxp3 shRNA2 and shRNA control to human colorectal cancer cells, which were named as Foxp3 shRNA1group, Foxp3 shRNA2 group, shRNA control group. At the same time, the non transfection group was established and transfection reagent was added into the non transfection group. After culturing 48 h, the expression level of Foxp3 in the cells was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), and the higher interference efficiency of shRNA was screened. Flow cytometry was used to detect the apoptosis of cells. Results The expression levels of Foxp3 in the adjacent tissues and colon cancer tissues were: 0.24±0.05, 0.78±0.08, and the difference was statistically significant (P=0.000, t=63.254). The expression level of Foxp3 in human colorectal cancer cells HCT116, SW480, RKO was higher than that of CCC-HIE-2, and the expression level of Foxp3 was from low to high: RKO, HCT116, SW480. Later selection SW480 continue to study. The expression level of Foxp3 protein in the transfected group, shRNA control group, Foxp3 shRNA1 group and Foxp3 shRNA2 group were: 0.92±0.05, 0.93±0.07, 0.21±0.04, 0.34±0.06. The expression level of mRNA was as follows: 1.00±0.07, 1.01±0.08, 0.53±0.06, 0.79±0.08. Subsequent Foxp3 shRNA2 interference Foxp3 expression. The apoptosis rates of the non transfection group, shRNA control group and Foxp3 shRNA2 group were as follows: (13.21±3.12)%, (12.98±3.54)%, (42.01±6.72)%. The expression level of B cell lymphoma/lewkmia-2 (bcl-2) in Foxp3 group shRNA2 was 0.86±0.05, 0.85±0.06, 0.26±0.05. The expression level of cleaved cysteinyl aspartate specific proteinase 3 (Cleaved Caspase-3) was: 0.38±0.09, 0.39±0.08, 0.88±0.06. The expression level of p-signal transducers and activators of transcription 3 (STAT3) was as follows: 0.40±0.06, 0.39±0.04, 0.22±0.05. Conclusion Foxp3 was up-regulated in colorectal cancer tissues and cells. Interference of Foxp3 expression could promote apoptosis of human colorectal cancer cells. The mechanism may be related to STAT3 signaling pathway. Key words: Colorectal cancer; Apoptosis; Forkhead/winged helix transcripition factor 3; Signal transducers and activators of transcription 3 signaling pathway

Interleukin-9 and soluble tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid of nonsegmental vitiligo patients

Beneficial therapeutic effect of Chinese herbal Bushen formula on CHB patients with mildly elevated alanine aminotransferase by down-regulating CD4+CD25+T cells